Hydrolysis and decomposition of small peptides in marine environments

Date

2016-05-11

Authors

Liu, Shuting

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Abstract

Proteins and peptides are important labile organic matter supporting growth of microorganisms in seawater. Small peptides (<600 Da) are key intermediates linking protein degradation, nutrient regeneration and DON preservation. In this dissertation, hydrolysis and/or decomposition of small peptides in seawater are investigated from both chemistry and biology perspectives. From the chemistry perspective, a new HPLC-MS method was first developed to measure small peptides amended in seawater at nanomolar levels. This method offers an easy and quick protocol to measure peptide concentrations in seawater without desalting pretreatments and lowers the detection limit by two orders of magnitude over the traditional UV method. It provides an analytical foundation for the peptide detection. With this method, hydrolysis of plain peptide without fluorogenic tags and peptide analogs in seawater was compared to assess the reliability of using small plain peptides as proxies. While Lucifer Yellow Anhydride (LYA) tag did not influence peptide hydrolysis rates significantly in many cases, it did affect the peptide hydrolysis pathways and susceptibility of dipeptide bonds to enzymes. This result validates the advantages of using plain peptides to study peptide hydrolysis rates and pathways. Peptide hydrolysis pathways were evaluated further to quantify the relative roles of different peptidases in seawater. Incubations of peptides with different chemical structures demonstrated that aminopeptidases prefer to cleave N-terminal hydrophobic or basic amino acids rather than polar uncharged or acidic ones in peptides. From the biology perspective, as bacteria are major consumer of labile organic matter such as peptides, linking bacteria communities and peptide decomposition using DNA-stable isotope probing is considered to explore peptide decomposition mechanisms. Different bacterial taxa were involved in peptide utilization between the normoxic and hypoxic seawater in northern Gulf of Mexico, offering insights into the biological roles of bacteria in organic matter decomposition and hypoxia formation. The potential role of microbes other than bacteria, such as protists, in peptide decomposition was also evaluated using size-fractioned seawater incubations, highlighting the need to include relatively large-size microorganisms in microbial loop to understand C and N cycles in ocean. This study examined peptide hydrolysis and decomposition in terms of overall rates, difference of pathways between environments, and interactions with different microorganisms, extending from bulk analysis of peptide degradation rates to detailed mechanisms including enzyme functions and microbial linkages. The results offer insights into labile organic matter cycling, microbial ecology, and nutrient regeneration in seawater, and also open more questions about the factors controlling the hydrolysis and decomposition patterns of labile organic matter, microbial behavior and functions in biogeochemical processes, and DON preservation mechanisms.

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